In the previous funding period of this IRPG grant submitted with that of Jeffrey Milbrandt, we reported the discovery of neurotrophic factors neurturin, persephin, and artemin, which along with GDNF, constitute the GFL family of neurotrophic factors. We, and others, also discovered GPI-anchored co-receptors for neurturin and artemin. Among many other issues, we have addressed signal transduction of GFLs and provided evidence for a critical role for lipid rafts and the cytoplasmic tyrosine kinase C-src in mediating GFL action. In preliminary experiments we have discovered in sensory and sympathetic neurons a novel, non-GFL-mediated, maturation-dependent phosphorylation of the receptor kinase Ret. Surprisingly, this maturation-dependent phosphorylation, at least in sympathetic neurons, appears reliant upon nerve growth factor. We have also observed robust effects of GFLs on basal forebrain neurons in vitro and atrophic changes in basal forebrain neurons from aged neurturin-/- mice in vivo. We shall perform experiments (Aim 1) to understand the biochemical mechanism, cellular specificity, and generality of the ligand-independent, maturation-dependent phosphorylation of Ret. We shall do experiments (Aim 2) both in vitro and in vivo to assess potential biological significance of this phenomenon. We shall examine (Aim 3A) the structural/biochemical basis of the interaction of Ret with C-src and use cells from src-/- mice to assess critically the role C-src in mediating GFL actions and determine whether neuronal populations dependent on GFLs show neuronal loss and/or atrophy in src-/- mice (Aims 3B and 3C). We shall complete experiments (Aim 4) to define further the role and mechanisms of the interaction of the GFL-signaling apparatus and to determine the nature of the lipid-raft environment used by GPI-anchored co-receptors to activate Ret in response to GPL stimulation. We shall study (Aim 5) the role of the GFLs in basal forebrain neurons in vitro, examine their potential as neuroprotectants in vivo, and examine neurturin-/- mice for age-dependent neurodegeneration by anatomical, neurochemical, and behavioral criteria. Lastly, we shall continue to collaborate with the Milbrandt lab in the execution of the Aims in the companion IRPG grant, including analysis of the artemin- and persephin-knock-out animals.